Alcohol dehydrogenase coexisted solid-state electrochemiluminescence biosensor for detection of p53 gene

An alcohol dehydrogenase （ADH）-coexisted solidstate electrochemiluminescence （ECL） biosensor for sensitive detection of the p53 gene was developed. The electrode modified by multiwalled carbon nanotubes, Ru（bpy）]2＋3 and polypyrrole （ MWNTs-Ru （bpy） ]2＋3 -PPy ） was prepared to adsorb the ssDNA by electrostatic interactions. Then, the ssDNA recognized the gold nanoparticles （AuNPs）-labeled p53 gene and produced the AuNPs-dsDNA electrode with the AuNPs layer. The AuNPs layer adsorbed the ADH molecules for producing the ECL signal. Thus, the biosensor was based on coupling enzyme substrate reaction with solid-state ECL detection, and it displayed good sensitivity and specificity. The detection limit of the wild type p53 sequence （wtp53） is as low as 0. 1 pmol/L and the discrimination is up to 57. 1% between the wtp53 and the muted type p53 sequence （mtp53）. The amenability of this method to the analyses of p53 from normal and cancer cell lysates is demonstrated. The signal of wtp53 in the MGC-803 gastric cancer cell lysates turns out to be about 61.8% that of the wtp53 in the GES-1 normal gastric mucosal cell lysates, and the concentration of the wtp53 is found to decrease about 59 times. The method is highly complementary to enzyme-linked immunosorbent assay （ELISA）, and it holds promise for the diagnosis and management of cancer.

In Vitro and In Vivo Effects and Safety Assessment of Corn Peptides on Alcohol Dehydrogenase Activities

The in vitro and in vivo effects of corn peptides（CPs） prepared from corn gluten meal by proteolysis with an alkaline protease and fractions of CPs from Sephadex G-15 and G-10 columns on activities of alcohol dehydroge-nase（ADH） were studied. The results show that CPs and fraction 3 of CPs from Sephadex G-10 column enhance in vitro ADH activity. Furthermore, the in vitro accelerating effect of the fraction 3 of CPs on ADH activity was superior to that of glutathione, which was also found even in the presence of ADH inhibitor, such as pyrazole. In the in vivo experiments, the animals were fed with different dosages of CPs and with a dose of Chinese distilled spirit orally, and sacrificed for the measurement of ADH activity. In vivo experimental results indicate that CPS enhanced hepatic ADH activities. To test the safety of CPs as health food, 30 d feeding test was performed. No obvious toxic effects were detected in treated Wistar rats.

Identification and expression patterns of alcohol dehydrogenase genes involving in ester volatile biosynthesis in pear fruit

Alcohol dehydrogenase （ADH） catalyzes the interconversion of aldehydes and their corresponding alcohols, and is a key enzyme in volatile ester biosynthesis. However, little is known regarding ADH and ADH encoding genes （ADHs） in pear. We identified 8 ADHs in the pear＇s genome （PbrADHs） by multiple sequences alignment. The PbrADHs were highly ho- mologous in their coding regions, while were diversiform in structure. 9 introns were predicted in PbrADH3-PbrADH8, while 8 introns, generated through exon fusion and intron loss, were predicted in PbrADH1 and PbrADH2. To study the genetic regulation underlying aroma biogenesis in pear fruit, we determined the PbrADH transcripts, ADH activities and volatile contents of fruits during ripening stage for Nanguoli and Dangshansuli, two cultivars having different aroma characteristics. ADH activity was strongly associated with the transcription of ADH~ in the two cultivars during fruit ripening stage. The higher ester content paralleling to a higher ADH activity was detected in Nanguoli than in Dangshansuli, so it is induced that the lower ester content in Dangshansuli fruit may be the result of weak ADH activity. The present study revealed that total ADH activity and volatile ester production correlated with increased PbrADHstranscript levels. PbrADH6 may contribute to ADH activity catalyzing aldehyde reduction and ester formation in pear fruit.

Genetic polymorphisms in cytochrome P4502E1, alcohol and aldehyde dehydrogenases and the risk of esophageal squamous cell carcinoma in Gansu Chinese males

AIM:To evaluate the association between genetic polymorphisms in CYP2E1, ALDH2 and ADH1B and the risk of esophageal squamous cell carcinoma (ESCC) in a high risk area of Gansu Province, in Chinese males. METHODS: A case-control study was conducted to investigate the genetic polymorphisms of these enzymes (CYP2E1 *c1/*c2, ALDH2 *1/*2 and ADH1B *1/*1 genotypes). A total of 80 esophageal cancer cases and 480 controls were recruited. RESULTS: Compared with controls, cases had a greater prevalence of heavier alcohol consumption (53.8% vs 16.2%) and a higher proportion of alcohol drinkers with > 30 drink-years (28.8% vs 13.5%). Heavier alcohol consumption and alcohol drinking with > 30 drink- years increased the risk of ESCC, with ORs (95% CI) of 3.20 (1.32-9.65) and 1.68 (0.96-3.21). CYP2E1 (*c1/*c1), ALDH2 (*1/*2) and ADH1B (*1/*1) genotype frequencies were higher among patients with squamous cell carcinomas, at a level close to statistical significance (P = 0.014; P = 0.094; P = 0.0001 respectively). There were synergistic interactions among alcohol drinking and ALDH2, ADH1B and CYP2E1 genotypes. The risk of the ESCC in moderate-to-heavy drinkers with an inactive ALDH2 encoded by ALDH2 *1/*2 as well as ADH1B encoded by ADH1B *1/*1 and CYP2E1 encoded by CYP2E1 *c1/*c1 was higher than that in the never/rare-to-light drinkers with an active ALDH2 (*1/*1 genotype) as well as ADH1B (*1/*2 + *2/*2) and CYP2E1 (*c1/*c2 + *c2/*c2) genotypes, with a statistically significant difference; ORs (95% CI) of 8.58 (3.28-22.68), 27.12 (8.52-70.19) and 7.64 (2.82-11.31) respectively. The risk of the ESCC in moderate-to-heavy drinkers with ALDH2 (*1/*2) combined the ADH1B (*1/*1) genotype or ALDH2 (*1/*2) combined the CYP2E1 (*c1/*c1) genotype leads to synergistic interactions, higher than drinkers with ALDH2 (*1/*1) + ADH1B (*1/*2 + *2/*2), ALDH2 (*1/*1) + CYP2E1 (*c1/*c2 + *c2/*c2) respectively , ORs (95% CI) of 7.46 (3.28-18.32) and 6.82 (1.44-9.76) respectively. Individuals with the ADH1B combined the CYP2E1 genotype showed no synergistic interaction. CONCLUSION: In our study, we found that alcohol consumption and polymorphisms in the CYP2E1, ADH1B and ALDH2 genes are important risk factors for ESCC, and that there was a synergistic interaction among polymorphisms in the CYP2E1, ALDH2 and ADH1B genes and heavy alcohol drinking, in Chinese males living in Gansu Province, China.

Relationship between genetic polymorphisms of alcohol and aldehyde dehydrogenases and esophageal squamous cell carcinoma risk in males

AIM： To investigate the association between the genetic polymorphisms of ADH2 and ALDH2, lifetime alcohol consumption and esophageal cancer risk in the Taiwan Residents men. METHODS： Between August 2000 and June 2003, 134 pathologically-proven esophageal squamous cell carcinoma male patients and 237 male controls were recruited from Kaohsiung Medical University Hospital and Kaohsiung Veterans General Hospital in southern Taiwan. ADH2 and ALDH2 polymorphisms were genotyped using PCR-RFLP. RESULTS： Compared to those with ADH2＊2/＊2, individuals with ADH2＊1/＊2 and ADH2＊1/＊1 had 2.28- and 7.14-fold, respectively, increased risk of developing esophageal cancer （95%CI = 1.11-4.68 and 2.76-18.46） after adjusting for alcohol consumption and other covariates. The significant increased risk was also noted among subjects with ALDH2＊1/＊2 （adjusted OR （AOR） = 5.25, 95%CI = 2.47-11.19）, when compared to those with ALDH2＊1/＊1. The increased risk of esophageal cancer was made greater, when subjects carried both ADH2＊1/＊1 and ALDH2＊1/＊2, compared to those with ADH2＊1/＊2 or ADH2＊2/＊2 and ALDH2＊1/＊1 （AOR = 36.79,95%a = 9.36-144.65）. Furthhermore, we found a multipticative effect of lifetime alcoholic consumption and genotypes （ADH2 and ALDH2） on esophageal cancer risk. CONCLUSION： Our findings suggest that polymorphisms of ADH2 and ALDH2 can modify the influence of alcoholic consumption on esophageal cancer risk.

Polymorphisms of alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 and colorectal cancer risk in Chinese males

AIM: To evaluate the relationship between drinking and polymorphisms of alcohol dehydrogenase 2 (ADH2) and/or aldehyde dehydrogenase 2 (ALDH2) for risk of colorectal cancer (CRC) in Chinese males. METHODS: A case-control study was conducted in 190 cases and 223 population-based controls. ADH2 Arg47His (G-A) and ALDH2 Glu487Lys (G-A)genotypes were identified by PCR and denaturing high-performance liquid chromatography (DHPLC). Information on smoking and drinking was collected and odds ratio (OR) was estimated. RESULTS: The ADH2 A/A and ALDH2 G/G genotypes showed moderately increased CRC risk. The age- and smoking-adjusted OR for ADH2 A/A relative to G/A and G/G was 1.60 (95% CI=1.08-2.36), and the adjusted OR for ALDH2 G/G relative to G/A and A/A was 1.79 (95% CI=1.19-2.69). Signif icant interactions between ADH2, ALDH2 and drinking were observed. As compared to the subjects with ADH2 G and ALDH2 A alleles, those with ADH2 A/A and ALDH2 G/G genotypes had a signif icantly increased OR (3.05, 95% CI= 1.67-5.57). The OR for CRC among drinkers with the ADH2 A/A genotype was increased to 3.44 (95% CI= 1.84-6.42) compared with non-drinkers with the ADH2 G allele. The OR for CRC among drinkers with the ALDH2 G/G genotype was also increased to 2.70 (95% CI= 1.57-4.66) compared with non-drinkers with the ALDH2 A allele. CONCLUSION: Polymorphisms of the ADH2 and ALDH2 genes are significantly associated with CRC risk. There are also signifi cant gene-gene and gene- environment interactions between drinking and ADH2 and ALDH2 polymorphisms regarding CRC risk in Chinese males.

Selective Affinity Separation of Yeast Alcohol Dehydrogenase by Reverse Micelles with Unbound Triazine Dye

The reversed micelles were formed with cationic cetyltrimethylammonium bromide (CTAB) as surfactant and n-hexanol as cosolvent in the CTAB (50mmol.L-1)/hexanol (15% by volume)/hexane system. Cibacron Blue 3GA (CB) as an affinity ligand in the aqueous phase was directly introduced to the reversed micelles with electrostatic interaction between anionic CB and cationic surfactant. High molecular weight (Mr) protein, yeast alcohol dehydrogenase (YADH, Mr = 141000) from baker's yeast, has been purified using the affinity reversed micelles by the phase transfer method. Various parameters, such as CB concentration, pH and ionic strength, on YADH forward and backward transfer were studied. YADH can be transferred into and out from the reversed micelles under mild conditions (only by regulation of solution pH and salt concentration) with the successful recovery of most YADH activity. Both forward and backward extractions occurred when the aqueous phase pH＞pI with electrostatic attraction between YADH and CTAB. The recovery of YADH activity and purification factor have been improved with addition of a small amount of affinity CB. The recovery of YADH activity obtained was ～99% and the purification factor was about 4.0-fold after one cycle of full forward and backward extraction. The low ionic strength in the initial aqueous phase might be responsible for the YADH transfer into the reversed micellar phase.

The relationship between activities of hepatic and gastric alcohol dehydrogenase and occurrence of chronic alcoholic liver disease

Objective: To investigate the role of hepatic and gas-tric alcohol dehydrogenase (ADH) in different pa-thologic stages of alcoholic liver disease (ALD).Methods: Thirty-nine Wistar rats were divided ran-domly into two groups: model group (24) and con-trol group (15). The ALD model was established byinfusing alcohol into the stomach. After hepatic andgastric tissues had been stained by enzyme histo-cyto-chemistry assay, the activity varieties of hepatic andgastric ADH were observed by an optical micro-scope, and the activity alterations were also deter-mined by LUZEX-F image analysis as a semi-quanti-tative method.Results: The activity of hepatic ADH gradually in-creased, but that of gastric ADH gradually decreasedin the different pathologic stages of alcoholic liverdisease. There was a significant difference betweenthe model group and control group (P【0.05).Conclusions: Along with occurrence of ALD, the ac-tivity of hepatic ADH gradually increased, but thatof gastric ADH gradually decreased, showing thatthe activity alterations of hepatic and gastric ADHmay play an important role in the onset and develop-ment of ALD.

Alcohol Dehydrogenase Activity in Cultured Cells from Different Tobacco (Nicotiana tabacum L.) Varieties

The activity of alcohol dehydrogenase (ADH) in cultured cells of various tobacco was determined. It was found that significant differences existed in cells of different varieties cultured under normal conditions and as well after treated with exogenous ethanol. The ADH activity had positive relation with the ability of the cells to catabolize exogenous ethanol, indicating that the main function of the ADH in tobacco cells was in the direction of converting ethanol to acetaldehyde.

Achieving Enzyme Stability Using a Simple Fabrication Procedure： The Alcohol Dehydrogenase Example

The use of screen-printing biosensors has been updated in this article as a tool to analyze the electron transfer process involving redox proteins or enzymes. The aim of this research was to fabricate a simple apparatus which allowed the use of the enzymes （in the solid state） to maintain their stability. To prove this concept an enzyme in the solid state was mixed with the carbon ink and this mixture was used to print the working electrode. We choose as proving the alcohol dehydrogenase. The first reason is because it metabolizes the alcohol, which can be present in biological samples of blood, saliva and urine and also in the beverage; the second is that this enzyme is still a challenge to electrochemistry due to having lower stability in sensors. The results show that in this device the enzyme was active and stable during all the experiments and in the experimental conditions that could catalyze the ethanol to acetaldehyde. These devices have the advantage of being disposable, cheap and are easy to fabricate. And also, they do not need expensive tools to be fabricated, they only need 2μL of electrolyte or sample, and they need lower amounts of enzyme to permit electrochemical studies.

Aldehyde dehydrogenase 2 overexpression inhibits neuronal apoptosis after spinal cord ischemia/reperfusion injury

Aldehyde dehydrogenase 2（ALDH2）is an important factor in inhibiting oxidative stress and has been shown to protect against renal ischemia/reperfusion injury.Therefore,we hypothesized that ALDH_2 could reduce spinal cord ischemia/reperfusion injury.Spinal cord ischemia/reperfusion injury was induced in rats using the modified Zivin＇s method of clamping the abdominal aorta.After successful model establishment,the agonist group was administered a daily consumption of 2.5%alcohol.At 7 days post-surgery,the Basso,Beattie,and Bresnahan score significantly increased in the agonist group compared with the spinal cord ischemia/reperfusion injury group.ALDH_2expression also significantly increased and the number of apoptotic cells significantly decreased in the agonist group than in the spinal cord ischemia/reperfusion injury group.Correlation analysis revealed that ALDH_2 expression negatively correlated with the percentage of TUNEL-positive cells（r=-0.485,P〈0.01）.In summary,increased ALDH_2 expression protected the rat spinal cord against ischemia/reperfusion injury by inhibiting apoptosis.

Association of genetic polymorphisms of aldehyde dehydrogenase-2 with esophageal squamous cell dysplasia

AIM:To demonstrate the possible associations between genetic polymorphisms of aldehyde dehydrogenase-2(ALDH2) and esophageal squamous cell dysplasia(ESCD).METHODS:All participants came from an area of high incidence of esophageal cancer and underwent an endoscopic staining examination;biopsies were taken from a non-staining area of the mucosa and diagnosed by histopathology.Based on the examinations,the subjects were divided into the control group with normal esophageal squamous epithelial cells and the ESCD group.ALDH2 genotypes of 396 cases were determined including 184 ESCD cases and 212 controls.The odds ratio(OR) and 95% confidence intervals(95% CI) were calculated by binary logistic regression models.RESULTS:The distribution of ALDH2 genotypes showed significant differences between the two groups.The adjustment factors were gender and age in the logistic regression models.Compared with 2*2/2*2 genotype,2*1/2*1 genotype was found to be a risk factor for ESCD,and the OR(95% CI) was 4.50(2.21-9.19).There were significant correlations between ALDH2 genotypes and alcohol drinking/smoking/history of esophageal cancer.CONCLUSION:The ALDH2 polymorphism is significantly associated with ESCD.

Efficient conversion of aromatic and phenylpropanoid alcohols to acids by the cascade biocatalysis of alcohol and aldehyde dehydrogenases

Benzyl and phenylpropanoid acids are widely used in organic synthesis of fine chemicals,such as pharmaceuticals and condiments.However,biocatalysis of these acids has received less attention than chemical synthesis.One of the main challenges for biological production is the limited availability of alcohol dehydrogenases and aldehyde dehydrogenases.Environmental microorganisms are potential sources of these enzymes.In this study,129 alcohol dehydrogenases and 42 aldehyde dehydrogenases from Corynebacterium glutamicum,Pseudomonas aeruginosa,and Bacillus subtilis were identified and explored with various benzyl and phenylpropanoid alcohol and aldehyde substrates,among which four alcohol dehydrogenases and four aldehyde dehydrogenases with broad substrate specificity and high catalytic activity were obtained.Moreover,a cascade whole-cell catalytic system including ADH-90,ALDH-40,and the NAD(P)H oxidase LreNox was established,which showed high efficiency in converting cinnamyl alcohol and p-methylbenzyl alcohol into the respective carboxylic acids.Remarkably,this biocatalytic system can be easily scaled up to gram-level production,facilitating preparation purposes.

A novel alcohol dehydrogenase in the hyperthermophilic crenarchaeon Hyperthermus butylicus

Hyperthermus butylicus is a hyperthermophilic crenarchaeon that produces 1-butanol as an end product.A thermostable alcohol dehydrogenase(ADH)must be present in H.butylicus to act as the key enzyme responsible for this production;however,the gene that encodes the ADH has not yet been identified.A novel ADH,HbADH2,was purified from a cell-free extract of H.butylicus,and its characteristics were determined.The gene that encodes HbADH2 was demonstrated to be HBUT_RS04850 and annotated as a hypothetical protein in H.butylicus.HbADH2 was found to be a primary-secondary ADH capable of using a wide range of substrates,including butyraldehyde and butanol.Butyraldehyde had the highest specificity constant,calculated as k_(cat)/K_(m),with kcat and apparent K_(m) values of 8.00±0.22 s^(-1) and 0.59±0.07 mM,respectively.The apparent Km values for other substrates,including ethanol,1-propanol,2-propanol,butanol,acetaldehyde,propanal,and acetone,were 4.36±0.42,4.69±0.41,3.74±0.46,2.44±0.30,1.27±0.18,1.55±0.20,and 0.68±0.04 mM,respectively.The optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were 6.0 and 9.0,respectively,while the optimal temperature was higher than 90°C due to the increase in enzymatic activity from 60℃ to 90℃.Based on its substrate specificity,enzyme kinetics,and thermostability,HbADH2 may be the ADH that catalyzes the production of 1-butanol in H.butylicus.The putative conserved motif sites for NAD(P)^(+)and iron binding were identified by aligning HbADH2 with previously characterized Fe-containing ADHs.

Meta-analysis reveals up-regulation of cholesterol processes in non-alcoholic and down-regulation in alcoholic fatty liver disease

AIM To compare transcriptomes of non-alcoholic fatty liver disease(NAFLD) and alcoholic liver disease(ALD) in a meta-analysis of liver biopsies.METHODS Employing transcriptome data from patient liver biopsies retrieved from several public repositories we performed a meta-analysis comparing ALD and NAFLD.RESULTS We observed predominating commonalities at the transcriptome level between ALD and NAFLD,most prominently numerous down-regulated metabolic pathways and cytochrome-related pathways and a few up-regulated pathways which include ECM-receptor interaction,phagosome and lysosome.However some pathways were regulated in opposite directions in ALD and NAFLD,for example,glycolysis was down-regulated in ALD and up-regulated in NAFLD.Interestingly,we found rate-limiting genes such as HMGCR,SQLE and CYP7A1 which are associated with cholesterol processes adversely regulated between ALD(down-regulated) and NAFLD(up-regulated).We propose that similar phenotypes in both diseases may be due to a lower level of the enzyme CYP7A1 compared to the cholesterol synthesis enzymes HMGCR and SQLE.Additionally,we provide a compendium of comparative KEGG pathways regulation in ALD and NAFLD.CONCLUSION Our finding of adversely regulated cholesterol processes in ALD and NAFLD draws the focus to regulation of cholesterol secretion into bile.Thus,it will be interesting to further investigate CYP7A1-mediated cholesterol secretion into bile-also as possible drug targets.The list of potential novel biomarkers may assist differential diagnosis of ALD and NAFLD.

Positive effect of ethanol-induced Lactococcus lactis on alcohol metabolism in mice

It is well known that exposure to environmental stresses could enhance the adaptability of bacteria and up-regulate the expression of a variety of oxidative stress-related genes and antioxidant enzymes.It is unclear whether the adaptability of microorganisms formed naturally in special environments could transfer to other organisms.The study aimed to evaluate the effects of untreated and ethanol-induced Lactococcus lactis intracellular extracts(U-IE and E-IE)on alcohol metabolism in mice.The positive effects of E-IE on alcohol metabolism in mice were revealed by the enhanced latency of loss of righting reflex(LORR),the reduced duration of LORR,the decrease of blood alcohol concentration,as well as the elevation of alcohol dehydrogenase(ADH)activities in the stomach and liver tissues.Furthermore,the potential benefits of E-IE on the liver were evaluated by biochemical parameters including the activities of serum transaminase,the levels of antioxidant enzymes,and the pathological changes of liver tissue.The present work put forward a new point that appropriate ethanol stress could enhance the intracellular ADH activity of L.lactis,and its intracellular extracts could continue to enhance alcohol metabolism in mice.

Aqueous Components of Tomato Accelerate Alcohol Metabolism by Increasing Pyruvate Level

Consumption of food while drinking alcohol has been suggested to play important roles in alleviating the physiological and pharmacological influences of alcohol. Vegetables are believed to provide health benefits, but there is little evidence for their influence on the effects of alcohol consumption. The present study aimed to investigate the effect of a common vegetable, tomato, on alcohol metabolism. In a randomized, controlled, crossover study with12 Japanese healthy men aged between 24 and 56 years, drinking tomato juice containing 5% (v/v) alcohol (TJAlc) significantly attenuated the elevation of blood ethanol level and subsequently increased the level of acetate compared with a water-based alcoholic beverage with an equal dose of alcohol (0.4 g/kg body weight). Significantly higher levels of blood pyruvate and lactate were also observed in subjects who had consumed TJAlc compared with those consuming the water-based beverage. Additionally, a biphasic alcohol effects scale method showed that subjective feelings for alcohol-induced stimulant effects were significantly enhanced by drinking TJAlc. Animal experiments using male Sprague Dawleyrats suggested that the effect on blood biomarkers was attributable to the serum fraction of tomato (TS), which largely consisted of aqueous compounds, but not lipophilic compounds such as the carotenoid lycopene. Furthermore, it was suggested the TS possibly included potent compound(s) in addition to alanine, glutamine, and citric acid, all of which have previously been reported to affect alcohol metabolism. Administration of TS clearly increased the activity of NAD (H)-dependent enzymes such as lactate-(LDH), alcohol-, and aldehyde-dehydrogenase in rat liver cytosols. These findings suggest that aqueous compound(s) in tomato promote alcohol metabolism, probably through increasing pyruvate level, enhancing LDH activity, and improving the ratio of NAD to NADH.

CAFs促进ADH1B甲基化对卵巢癌细胞增殖及侵袭的影响

目的 探讨肿瘤相关成纤维细胞(CAFs)分泌的IL-6对卵巢癌细胞增殖及侵袭的影响及机制。方法 收取新鲜离体上皮性卵巢癌及正常卵巢上皮组织,分离纯化获得CAFs及正常卵巢成纤维细胞(NFs);蛋白质印迹和免疫荧光实验检测上皮细胞和成纤维细胞标志物α-平滑肌动蛋白(α-SMA)、上皮型钙黏附素(E-cadherin)表达;收集CAFs和NFs培养上清液与卵巢癌SKOV3细胞建立间接共培养体系,细胞分为SKOV3单独培养(SKOV3)组、SKOV3与NFs上清液(NFs)组及SKOV3与CAFs上清液(CAFs)组;细胞免疫组化检测SKOV3细胞共CAFs及NFs上清液培养后乙醇脱氢酶1B(ADH1B)表达;甲基化特异性PCR (MSP)、逆转录实时荧光定量PCR(RT-qPCR)、酶联免疫吸附试验(ELISA)及蛋白质印迹实验分别检测甲基化抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-dC)干预前后各组细胞ADH1B mRNA表达及甲基化状态、信号转导和激活因子3(STAT3)蛋白磷酸化水平;细胞计数试剂盒8(CCK-8)法及Transwell实验分别检测IL-6抑制剂LMT-286及重组IL-6 (rhIL-6)对细胞增殖及侵袭能力的影响。结果 肿瘤成纤维细胞中高表达α-SMA,极低表达E-cadherin;相比较SKOV3组及NFs组,CAFs组ADH1B mRNA及蛋白表达明显下调,同时CAFs组细胞上清液中IL-6水平较SKOV3组及NFs组明显升高;5-Aza-dC作用后,ADH1B甲基化部分逆转;三组细胞ADH1B mRNA和蛋白表达均增加,CAFs组STAT3磷酸化水平下降;LMT-286及rhIL-6干预均仅抑制或促进CAFs组细胞增殖和侵袭,而SKOV3组和NFs组无明显改变。结论 CAFs通过IL-6/STAT3信号通路增强ADH1B甲基化促进卵巢癌细胞增殖和侵袭。

不同诱导物对白地霉中高级醇脱氢酶的影响

化学或物理因子作为诱导物,能够直接或间接影响菌体基因转录表达。为探索高级醇底物对白地霉的诱导效果,以分离自土壤的白地霉S12为对象,采用五种高级醇(正丙醇、正丁醇、异丁醇、正己醇和异戊醇)作为诱导物,研究不同高级醇的诱导剂量、诱导时间对菌体S12降解不同高级醇活性及脱氢酶活力的影响。结果表明,当正己醇和异丁醇分别作为诱导剂时,胞内酶活力较高。正丙醇、正丁醇、异丁醇和正己醇作为诱导剂时,最适诱导时间均为6 h;而以异戊醇为诱导剂时,最佳诱导时间为12 h。以正丙醇和正己醇为诱导剂时,最适的诱导浓度为1.5 g/L;以正丁醇、异丁醇和异戊醇为诱导剂时,最适诱导浓度分别为1.0、0.5和2.5 g/L。NativePAGE电泳结果表明,以五种高级醇为诱导剂均能使菌体合成分子量为223 kDa左右的脱氢酶。其中,以正己醇为诱导剂时所得菌体同时降解多种高级醇的能力最强,其降解产物均为其相应的酸类和酯类物质。